Using electron microscopy radioautography, the deposition of intravenously administered iron in the duodenal epithelium was studied in normal mice, iron-overloaded and iron-deficient mice, and in mice with X-linked anemia (gene symbol sla) 4 and 24 hours after injection of 59Fe. The resolution of radioautography with 59Fe was determined with a line source and the distance from the hot line within which half of the grains fell (HD value) was 1650 A. In normal, iron-overloaded, and sla mice, radioiron was localized in the undifferentiated crypt cells at 4 hours and in the absorptive cells of the luminal half of the villi, at 24 hours. At both times, the vast majority of the grains was seen over the areas rich in free ribosomes and rough endoplasmic reticulum. In iron-deficient mice, grains were not found at either time. The amount of iron incorporated in the crypt cells was related to the size of the body iron stores. It is postulated that the amount of iron incorporated in the crypt cells is the result of interaction between uptake and return to the blood of the circulating iron. Once the crypt cells have differentiated into absorptive cells, the uptake and recirculation of iron from and to the blood would cease, leaving an amount of "message" iron which determines the absorptive cell's subsequent capacity for iron transfer to the plasma. In sla mice, in spite of tissue iron deficiency, the amount of iron deposited was similar to that of normal mice and markedly increased after treatment of the anemia. The accumulation of iron in the crypt cells may result from the decreased return to the blood of the incorporated circulating iron, due to the postulated deficient iron carrier mechanism in sla, or may be a consequence of increased avidity of the absorptive cells for circulating iron. In either event, the result would be that the absorptive cell receives an inappropriate message with resulting inappropriate absorption of iron.